Japanese encephalitis is due to an infection with the mosquito-borne Japanese encephalitis virus (JEV), a member of the Flavivirus genus in the Flaviviridae family (Go et al., 2014; Hubalek et al., 2014; Weaver and Barrett, 2004; Yun and Lee, 2014). JEV contains a positive single-stranded RNA genome encoding a polyprotein that is processed into three structural proteins, the capsid (C), the precursor of membrane (prM) and the envelope (E), and seven nonstructural proteins NS1 to NS5 (Yun and Lee, 2014). Viral assembly occurs in the lumen of endoplasmic reticulum membrane where the nucleocapsids associate with the heterodimers prME to form immature JEV virion. The latter transits through the secretory pathway, where the virion is matured through cleavage of prM into the membrane (M) protein by furin in the trans-Golgi (Yun and Lee, 2014). Additionally, like other Flaviviruses, JEV produces Virus-Like Particles (VLPs), which are assembled solely from prM and E proteins, and undergo the same maturation process as genuine viral particles (Kuwahara and Konishi, 2010). These VLPs can be produced in the absence of any other viral component and display similar biological activity as genuine virions (Kuwahara and Konishi, 2010).
JEV is usually maintained in an enzootic cycle between Culex tritaeniorhynchus mosquitoes and amplifying vertebrate hosts, such as waterbirds and domestic swine (Go et al., 2014; Hubalek et al., 2014; Impoinvil et al., 2013). Horses and humans are thought to be dead-end hosts, since they do not develop a level of viremia sufficient to infect mosquitoes (Impoinvil et al., 2013). In the past decades, there has been an expansion of JEV geographic distribution in Asia and possible introduction of JEV in Europe has been recently documented (Campbell et al., 2011; Zeller 2012).
Phylogenetic studies based on the viral envelope protein sequences allow the division of JEV strains into genotypes G1 to G5 (Gao et al., 2014; Hubalek et al., 2014; Le Flohic et al., 2013; Schuh et al., 2013; Solomon et al., 2003; Weaver and Barrett, 2004). Initially, most of the circulating strains of JEV belong to G3 and were at the origin of major epidemics in Southeast Asian countries. Recently a shift in prevalence from JEV G3 to G1 has been observed in several Asian countries, while some strains of JEV G5 have been occasionally isolated in China and South Korea (Gao et al., 2013; Le Flohic et al., 2013; Li et al., 2014; Pan et al., 2011; Schuh et al., 2014; Takhampunya et al., 2011).
JEV is the etiologic agent of the most important viral encephalitis of medical interest in Asia, with an incidence of 50,000 cases and about 10,000 deaths per year (Campbell et al., 2011; Go et al., 2014; Yun and Lee, 2014). About 20 to 30% of the symptomatic human cases are fatal, while 30 to 50% of no lethal cases can develop long-term neurologic sequelae. No antiviral treatment is available for JE disease. Vaccines against JEV are currently available to humans and for some animals such as horses and swine: those are inactivated mouse brain-derived, inactivated cell culture derived, live-attenuated and live-attenuated chimeric yellow fever virus-JEV vaccines (Bonaparte et al., 2014; Dubischar-Kastner and Kanesan-Thasna, 2012; Erra et al., 2013; Fan et al., 2013; Halstead and Thomas, 2011; Impoinvil et al., 2013; Ishikawa et al., 2014; Marks et al., 2012; Song et al., 2012; Yang et al., 2014; Yun and Lee, 2014). However, some of them lack of long-term immunity and live-attenuated vaccine strains carry a possible risk of reversion to virulence (Yun and Lee, 2014). Also the cost effectiveness of JEV vaccines is considered as a major obstacle (Impoinvil et al., 2013).
Lentiviral vectors represent a novel and attractive platform for gene-based immunization. The ability of lentiviral vectors to efficiently transduce non-dividing dendritic cells (DCs) allows a prolonged antigen presentation through the endogenous pathway, which in turns translates into the induction of strong, multi-epitopic and long lasting humoral as well as cellular immune responses. Consequently, an increasing number of pre-clinical studies show a great vaccine efficacy of lentiviral vectors in both infectious diseases and anti-tumor vaccination fields (Beignon et al., 2009; Di Nunzio et al., 2012; Fontana et al., 2014; Grasso et al., 2013; Hu et al., 2011; Sakuma et al., 2012). The inventors previously demonstrated that both integrative and non-integrative lentiviral vectors are promising vaccination vectors against arboviruses such as West Nile virus (WNV) that belongs to the JE serocomplex of Flavivirus genus (Coutant et al., 2008; Iglesias et al., 2006). These reports represented the first demonstration of the ability of lentiviral vectors for eliciting a protective antibody response against an infectious pathogen. Indeed immunization with a single minute dose of recombinant lentiviral TRIP vectors that express the soluble form of WNV E protein confers a robust sterilizing protection against a lethal challenge with WNV in mice (Coutant et al., 2008; Iglesias et al., 2006). Humoral immunity plays a pivotal role in protecting from JEV infection (Konishi, 2013; Dubischar-Kastner and Kanesan-Thasna, 2012) and consequently, the elicitation of protective antibody response is critical in the development of safe JEV vaccines (Larena et al., 2013).
International patent application WO2005/111221 relates to a recombinant lentiviral vector for expression of a protein of a Flaviviridae and to its applications as a vaccine. In particular it describes the use of a recombinant lentiviral vector comprising a polynucleotide fragment encoding at least one protein of a virus of the family Flaviviridae or an immunogenic peptide of at least 8 amino acids of said protein, for preparing an immunogenic composition intended for the prevention and/or the treatment of a Flaviviridae infection in a sensitive species.
International patent application WO2007/052165 relates to the use of a lentiviral vector comprising a heterologous nucleic acid encoding an antigen, and wherein expression of the antigen in a cell of an animal elicits a humoral response in said animal, for the preparation of a medicament able to produce antibodies when administered to said animal. For example, expression of the antigen induces protective immunity against a flavivirus, i.e. WNV.
International patent application WO2009/019612 relates to lentiviral gene transfer vectors and to their medicinal application. These vectors may be used to elicit an immune response to prevent or to treat a pathogenic state, including virus infections, parasite and bacterial infections or cancers. Said lentiviral vector can comprise a polynucleotide encoding at least one antigenic polypeptide derived from a flavivirus, for example from JEV.
International patent application WO2005/065707 relates to two recombinant adenoviruses (RAds), namely RAdEa expressing prM and the membrane-anchored E protein (Ea) of JEV, and RAdEs expressing prM and the secretory E protein (Es) of JEV. Plasmids pMEa and pMEs containing the cDNAs encoding prM and said Ea or Es of JEV have been described by Kaur et al. (2002).
Having considered the persistent need for a vaccine providing a protective humoral immune response against a JEV infection including against multiple JEV genotypes, the inventors have designed a novel lentivirus vector expressing JEV selected proteins that proved to elicit a protective immune response against one or more JEV of different genotypes. The obtained results show that recombinant TRIP vectors expressing JEV prM and E proteins may prime and boost antigen-specific humoral broadly neutralizing responses in vaccinated mice.